Patent application title: LASER SCANNING MICROSCOPE

Abstract:

In a laser scanning microscope comprising an infrared pulse laser; an
objective lens focusing an infrared light from the infrared pulse laser
on a sample; a condenser lens disposed on an opposite side of the
objective lens across the sample for collecting an observation light that
is generated by a nonlinear optical effect and has a wavelength shorter
than a wavelength of the infrared light; a visible light detector
detecting the observation light collected by the condenser lens, an IR
partial transmission filter having partially-modified transmission
characteristics for the infrared light is disposed near a front focal
position of the condenser lens, and an infrared light detector detecting,
through the IR partial transmission filter, a transmitted light from the
sample collected by the condenser lens, is provided.

Claims:

1. A laser scanning microscope comprising:an infrared pulse laser;an
objective lens focusing an infrared light from the infrared pulse laser
on a sample;a condenser lens disposed on an opposite side of the
objective lens across the sample for collecting an observation light that
is generated by a nonlinear optical effect and has a wavelength shorter
than a wavelength of the infrared light;a visible light detector
detecting the observation light collected by the condenser lens;an IR
partial transmission filter that is disposed near a front focal position
of the condenser lens arid has partially-modified transmission
characteristics with respect to the infrared light; andan infrared light
detector detecting, through the IR partial transmission filter, a
transmitted light from the sample collected by the condenser lens.

3. A laser scanning microscope comprising:an infrared pulse laser;an
objective lens focusing an infrared light from the infrared pulse laser
on a sample;a condenser lens disposed on an opposite side of the
objective lens across the sample for collecting an observation light that
is generated by a nonlinear optical effect and has a wavelength shorter
than a wavelength of the infrared light;a visible light detector
detecting the observation light collected by the condenser lens;an IR
partial transmission filter that is disposed near a front focal position
of the condenser lens and has partially-modified transmission
characteristics with respect to the infrared light;an infrared light
source illuminating the sample through the condenser lens and the IR
partial transmission filter; andan infrared light detector detecting a
transmitted light from the sample collected by the objective lens.

5. The laser scanning microscope according to claim 1, whereinthe IR
partial transmission filter is configured so thatthe visible light is
passed through an entire area of an effective flux, andthe infrared light
is passed through an area eccentric to a center of an effective flux.

6. The laser scanning microscope according to claim 3, whereinthe IR
partial transmission filter is configured so thatthe visible light is
passed through an entire area of an effective flux, andthe infrared light
is passed through an area eccentric to a center of an effective flux.

7. The laser scanning microscope according to claim 1, whereinthe IR
partial transmission filter is composed by a first sector-shaped area and
a second sector-shaped area,the first sector-shaped area transmitting the
visible light but not transmitting the infrared light, andthe second
sector-shaped area transmitting both of the infrared light and the
visible light.

8. The laser scanning microscope according to claim 3, whereinthe IR
partial transmission filter is composed by a first sector-shaped area and
a second sector-shaped area,the first sector-shaped area transmitting the
visible light but not transmitting the infrared light, andthe second
sector-shaped area transmitting both of the infrared light and the
visible light.

9. The laser scanning microscope according to claim 1, whereinthe IR
partial transmission filter is composed by overlapping a first optical
filter and a second optical filter, andillumination states of oblique
illumination are changed by changing relative positions of the first
optical filter and the second optical filter.

10. A laser scanning microscope according to claim 3, whereinthe IR
partial transmission filter is composed by overlapping a first optical
filter and a second optical filter, andillumination states of oblique
illumination are changed by changing relative positions of the first
optical filter and the second optical filter.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]This application claims benefit of Japanese Application No.
2007-282653, filed Oct. 31, 2007, the contents of which are incorporated
by this reference.

BACKGROUND OF THE INVENTION

[0002]1. Field of the Invention

[0003]The present invention relates to the technical field of microscopes.

[0004]2. Description of the Related Art

[0005]A fluorescence observation method using multiphoton excitation has
been known as a method of fluorescence observation with a microscope.

[0006]With the multiphoton excitation, a fluorescent material is
irradiated with light beams having a wavelength corresponding
approximately to the integral multiple of the absorption wavelength
simultaneously, inducing an excitation phenomenon that is equivalent to
the excitation phenomenon generated by the original absorption
wavelength. The multiphoton excitation phenomenon is called a nonlinear
phenomenon, which occurs with a rate proportional to the square of the
intensity of the excitation light in the case of, for example, two-photon
excitation.

[0007]Meanwhile, the light density of an excitation light focused by an
objective lens of a microscope decreases in inverse proportion to the
square of the distance from the focal plane. In other words, the
multiphoton excitation phenomenon in a microscope occurs only in the area
very close to the focal point, and the fluorescence is emitted only from
that area.

[0008]Thanks to the above characteristics of the multiphoton excitation, a
multiphoton-excitation laser scanning microscope does not require a
confocal pinhole on the detection side that is used in a normal confocal
microscope for shutting out the emission of fluorescence in areas other
than the focal plane. The multiphoton excitation also has an advantage
that the discoloration by the fluorescence in a sample can be suppressed,
since the excitation phenomenon occurs only on the focal plane.

[0009]Meanwhile, since the excitation light used for the multiphoton
excitation has a longer wavelength than usual, it generally becomes a
light beam in the infrared domain. Generally, a light with a longer
wavelength is less prone to scatter (Rayleigh scatter). Therefore, the
excitation light used in the multiphoton excitation has a characteristic
that it reaches deeper in a specimen having a scattering property, such
as a living specimen. This means that the use of multiphoton excitation
enables the observation into a deep area in a living body that could not
be attained with a normal visible light.

[0010]Thus, the fluorescence observation utilizing the multiphoton
excitation in a microscope has now become very effective.

[0011]Similarly, in a microscope utilizing a Second-Harmonic Generation
(SHG), a light having a half wavelength of the irradiation light is
detected. Therefore, an SHG microscope also has advantages such as less
influence from the Rayleigh scatter and less light invasion caused by the
light to the sample.

[0012]The observation of a specimen using the above-described microscopes
often involves a preparation of the specimen in advance, using another
observation method. Particularly, when observing a specimen using the
patch-clamp method, an electrode needs to be disposed accurately on a
specific position in the specimen. Conventionally, in such a case, the
multiphoton-excitation observation and SHG observation have been carried
out, after attaching a patch clamp using observation methods utilizing
differential interference contrast (DIC) or oblique illumination.

[0013]However, the preparation of a specimen using the DIC and oblique
illumination has a significant problem.

[0014]The DIC observation requires a Nomarski prism or a Wollaston prism
to be disposed on the image side of the objective lens. However, the
laser entering the objective lens is to produce spots while it is spilt
by the prism into two light fluxes that are slightly shifted sideways on
the sample position, causing the degradation of resolution and decrease
in brightness. For this reason, with the observation using the laser,
these prisms need to be removed from the light path, and they need to be
inserted into the path, with the DIC observation using a transmitted
illumination light. However, when carrying out observation using the
patch-clamp method, there have been problems such as the slight shake
generated by these operations causing the detachment of the electrode.

[0015]In addition, transmitted-light DIC using the laser requires a
linearly-polarized light with a high extinction ratio. However, the
extinction ratio of the laser itself is low, and the optical system on
the way to the objective lens breaks its polarization. Therefore, the
light needs to be transmitted through a polarizer. Meanwhile, in the
nonlinear microscopy observation method, the excitation efficiency is
proportional to an n-th power of the laser intensity. Therefore, the loss
of light intensity due to the polarizer causes decreases in the detection
sensitivity and in the depth limitation in deep observation of a
scattering substance.

[0016]Meanwhile, the contrast method using oblique illumination involves
the oblique illumination of a transmitted-illumination light, the angle
being provided by disposing a slit on a front focal position of the
condenser lens. For this reason, the slit needs to be removed from the
optical path, when performing the multiphoton-excitation observation or
the SHG observation. This also leads to problems such as the slight shake
generated by the insertion and removal of the slit causing the detachment
of the electrode.

SUMMARY OF THE INVENTION

[0017]An embodiment of the present invention is a laser scanning
microscope comprising an infrared pulse laser; an objective lens focusing
an infrared light from the infrared pulse laser on a sample; a condenser
lens disposed on an opposite side of the objective lens across the sample
for collecting an observation light that is generated by a nonlinear
optical effect and has a wavelength shorter than a wavelength of the
infrared light; a visible light detector detecting the observation light
collected by the condenser lens, in which an IR partial transmission
filter having partially-modified transmission characteristics with
respect to the infrared light is disposed near a front focal position of
the condenser lens, and an infrared light detector detecting, through the
IR partial transmission filter, a transmitted light from the sample
collected by the condenser lens, is provided.

[0018]Another embodiment of the present invention is a laser scanning
microscope comprising an infrared pulse laser; an objective lens focusing
an infrared light from the infrared pulse laser on a sample; a condenser
lens disposed on an opposite side of the objective lens across the sample
for collecting an observation light that is generated by a nonlinear
optical effect and has a wavelength shorter than a wavelength of the
infrared light; a visible light detector detecting the observation light
collected by the condenser lens, in which an IR partial transmission
filter having partially-modified transmission characteristics with
respect to the infrared light is disposed near a front focal position of
the condenser lens, and an infrared light source illuminating the sample
through the condenser lens and the IR partial transmission filter, and an
infrared light detector detecting a transmitted light from the sample
collected by the objective lens are disposed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]The present invention will be more apparent from the following
detailed description when the accompanying drawings are referenced.

[0020]FIG. 1 is a schematic diagram of a multiphoton-excitation laser
scanning microscope showing the first embodiment of the present
invention;

[0021]FIG. 2 is a schematic diagram of a multiphoton-excitation laser
scanning microscope showing the second embodiment of the present
invention;

[0022]FIG. 3A is a schematic diagram of an IR partial transmission filter
used for an embodiment of the present invention;

[0023]FIG. 3B is a schematic diagram showing a modified example of an IR
partial transmission filter used for an embodiment of the present
invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024]Hereinafter, embodiments of the present invention are described,
referring to the drawings. While the implementation of the present
invention is described herein using an example of a
multiphoton-excitation laser scanning microscope, the implementation is
not limited to the example, and the present invention may also be
implemented with an SHG microscope.

Embodiment 1

[0025]FIG. 1 is a schematic diagram illustrating an embodiment of a
multiphoton-excitation laser scanning microscope according to the present
invention. An infrared pulse laser 1 is generally used as the excitation
light source in a multiphoton-excitation laser scanning microscope. One
reason for using the pulse laser is that the photon density on the focal
plane can be effectively increased. Since the infrared pulse laser 1 is a
large apparatus, it is disposed outside the microscope, and an infrared
light (excitation light) from the infrared pulse laser 1 is directed to
the scanning unit 2. The configuration shown in FIG. 1 where the scanning
unit 2 is built into the microscope 3 is not a limitation, and the
scanning unit 2 may be disposed outside the microscope 3.

[0026]The infrared light emitted from the infrared pulse laser 1 is
directed to the scan unit 2, and is deflected by scanning means 4 such as
a galvano mirror disposed inside the scanning unit 2. Since the scanning
means is disposed on a conjugated position with respect to the pupil of
an objective lens, the deflected excitation light ultimately scans the
surface of a sample 8. After passing through the scanning means 4, the
excitation light enters the objective lens 7 via a pupil projection lens
5 and an imaging lens 6. As a result, the sample 8 is irradiated with the
infrared light (excitation light).

[0027]In the present embodiment, transmission detection is performed for a
fluorescence generated by the multiphoton excitation that is a nonlinear
phenomenon, (i.e., a result of a nonlinear optical effect). For this
purpose, the fluorescence emitted from the sample 8 is collected by a
condenser lens 9. Note that the condenser lens collects not only the
fluorescence emitted from the sample 8 but also the infrared light
(excitation light) passing through the sample 8.

[0028]In the present embodiment, an IR (infrared) partial transmission
filter 10 having partially-modified transmission characteristics (to be
described later using FIG. 3A and FIG. 3B) is disposed on the front focal
position of the condenser lens 9. A dichroic mirror 11 is disposed on a
subsequent stage of the IR partial transmission filter 10. A fluorescent
detection unit 12 is disposed in one of the light paths separated by the
dichroic mirror 11, and an infrared light detector 15 is disposed in the
other of the light paths. In this regard, since the fluorescence emitted
by the multiphoton excitation has a wavelength that is shorter than that
of the excitation light, the arrangement is made so that the light having
the shorter wavelength is directed to the fluorescent detection unit 12.
The present embodiment illustrates a configuration in which a
particularly short wavelength is reflected.

[0029]According to the configuration described above, the fluorescence
emitted from within the specimen by multiphoton excitation is collected
by the condenser lens 9, transmitted through the IR partial transmission
filter 10, reflected by the dichroic mirror 11, and directed to the
fluorescent detection unit 12.

[0030]The fluorescent detection unit 12 is equipped with an infrared light
cut filter 13 and a fluorescent detector 14, with which the fluorescent
detection unit 12 detects fluorescence with unneeded excitation light
being removed. In this regard, the fluorescent detector 14 is preferably
a photoelectron multiplier. In addition, instead of disposing a single
unit of the fluorescent detector 14, a plurality of the fluorescent
detectors 14 may be disposed to perform multi-channel detection. While
the fluorescent detection unit 12 is described in FIG. 1 as disposed
within the main body of the microscope 3, the present invention is not
limited to the arrangement. The light path may be directed in the
vertical direction (to plane of paper) in FIG. 1, and the fluorescent
detection unit 12 may be disposed outside the microscope.

[0031]Meanwhile, after passing through the specimen, the infrared light is
collected by the condenser lens 9, passes through the IR partial
transmission filter 10, passes through a dichroic mirror 11, and is
directed to an infrared light detected 15. In the present embodiment
shown in FIG. 1, the infrared light is configured to be directed, by
means of the light path that is normally used for the transmitted
illumination, to the infrared light detector 15 that is disposed in the
lamp housing part. In this regard, the infrared light detector 15 is
preferably a photoelectron multiplier.

[0032]The infrared light detector 15 is used in observing the figure of
the specimen, for the preparation of the specimen including the
disposition of a patch clamp and so on. Specifically, the sample 8 is
irradiated with a light from the infrared pulse laser 1 with its
intensity turned down, and the detection is performed for its transmitted
light. At this time, the IR partial transmission filter 10 that is
disposed on the front focal position of the condenser lens 9 partially
transmits the infrared light. More specifically, only the infrared light
that is emitted with an angle with respect to the sample 8 passes through
and reaches the dichroic mirror 11. Thus, for the infrared light
separated by the dichroic mirror 11 and directed to the infrared light
detector 15, the component of an observation light with oblique
illumination is detected.

[0033]Meanwhile, since the detected light in the multiphoton-excitation
observation is in the visible light range and is not subject to the
influence of the IR partial transmission filter 10, the
multiphoton-excitation observation can be carried out as normal. Thus,
according to the configuration described above, switching between the
multiphoton-excitation observation and the oblique illumination
observation can be performed without shifting the light paths or changing
the parts.

[0034]In addition, FIG. 1 shows mirrors 16 and 17 for controlling the
light path. The light path may also be controlled using a prism.

Embodiment 2

[0035]Hereinafter, the outline of the entire configuration of a
multiphoton-excitation laser scanning microscope according to another
embodiment is described referring to FIG. 2.

[0036]In the same manner as in Embodiment 1, an infrared pulse laser 1 is
generally used as the excitation light source. The infrared light emitted
from the infrared pulse laser 1 is directed to a scanning unit 2, passes
through scanning means 4, a pupil projection lens 5, an imaging lens 6,
and the like, and is irradiated on a sample 8 through an objective lens
7. In this regard, in the same manner as in Embodiment 1, the scanning
unit 2 may either be built into the microscope 3 or may be disposed
outside.

[0037]In the present embodiment, to detect the transmitted light as well
as in Embodiment 1, the fluorescence from the sample 8 is collected by a
condenser lens 9. After passing through the condenser lens 9, the
fluorescence is passed through an IR partial transmission filter 10 that
is disposed on the front focal position and has partially-modified
transmission characteristics (to be described in detail later using FIG.
3A and FIG. 3B), reflected by a dichroic mirror 11, and directed to a
fluorescent detection unit 12. While the fluorescent detection unit 12 is
described as disposed inside the main body of the microscope 3 as well as
in Embodiment 1, the implementation of the present invention not limited
to the arrangement.

[0038]The fluorescent detection unit 12 is equipped with an infrared light
cut filter 13 and a fluorescent detector 14, with which the fluorescent
detection unit 12 detects fluorescence with unneeded excitation light
being removed. In this regard, the fluorescent detector 14 is preferably
a photo electron multiplier. In addition, instead of disposing a single
unit of the fluorescent detector 14, a plurality of the fluorescent
detectors 14 may be disposed to perform multi-channel detection.

[0039]In the present embodiment, a fluorescent light detection unit 12 is
disposed in one of the light paths separated by the dichroic mirror 11,
and an infrared light source 18 is disposed in the other of the light
paths. The light source may be a light source using an infrared
radiation, such as a halogen lamp, instead of a pulse laser light source.
The example in FIG. 2 shows a configuration in which the infrared light
source 18 is disposed in a normal lamp housing position.

[0040]The infrared light emitted from the infrared light source 18 passes
through the dichroic mirror 11, and its light flux is limited by the IR
partial transmission filter 10. Since the IR partial transmission filter
10 is disposed on the front focal position of the condenser lens 9, the
infrared light is applied on the sample 8 as oblique illumination after
passing through the IR partial transmission filter 10.

[0041]The transmitted light from the sample 8 having been applied with the
oblique illumination is collected by the objective lens 7, and separated
for the excitation path (the light path towards the infrared pulse laser)
by a dichroic mirror 19, and imaged by an infrared light imaging device
20 that is an infrared light detector. In this regard, the infrared light
imaging device 20 is preferably a two-dimensional imaging device such as
an infrared CCD. In other words, an imaging lens not shown in the drawing
is disposed between the dichroic mirror 19 and the infrared light imaging
device 20, and the sample 8 and the infrared light imaging device 20 are
disposed at optically-conjugated positions.

[0042]As well as Embodiment 1, the embodiment described above enables
switching between the multiphoton-excitation observation and the oblique
illumination observation without shifting the light paths or changing
parts.

[0043]Hereinafter, the embodiments of the IR partial transmission filter
used in Embodiments 1 and 2 above are described, in reference to FIG. 3A
and FIG. 3B.

[0044]FIG. 3A shows an example of an IR partial transmission filter 10 in
the simplest form used for the implementation of the present invention.
This example shows a configuration in which the obtuse sector-shaped area
shown with the checkerboard pattern transmits a visible light but does
not transmit an infrared light. Meanwhile, the remaining acute-angled
sector-shaped area transmits both the infrared light and the visible
light. In other words, the IR partial filter 10 transmits the visible
light in the entire area that an effective flux enters. Meanwhile, the
infrared light is transmitted only in a partial area in which the
effective flux enters, more specifically, in a partial area that is
eccentric to the center of the effective flux. The oblique illumination
is realized by partially transmitting the infrared light according to the
configuration.

[0045]FIG. 3B shows another example of the IR partial transmission filter
10 used for the implementation of the present invention. In the example,
the IR partial filter is composed by overlapping two IR partial
transmission filters, i.e., a first IR partial transmission filter 21 and
a second IR partial filter 22. For example, as shown in FIG. 3B, the
first IR partial transmission filter 21 is provided with a sector-shaped
area that transmits both an infrared light and a visible light. On the
other hand, the second IR partial transmission filter 22 is provided with
a comma shaped area that transmits the visible light but does not
transmit the infrared light. By overlapping the first optical filter and
the second optical filter such as the first IR partial filter 21 and the
second IR partial filter 22 having different transmission characteristics
with respect to the infrared light and by adjusting their angles, the
illumination states of the oblique illumination such as the angle and
illumination intensity can be adjusted.

[0046]Meanwhile, embodiments of the IR partial transmission filter are not
limited to the above examples. For example, the area that transmits the
infrared light may take various shapes, such as a rectangular shape and
an arch shape. It is preferable that the variation of the relative
positions of the two IR partial transmission filters is adjusted in
accordance with the shape of the area that transmits the infrared light.
For example, when the shape of the area that transmits the infrared light
is rectangular, it is preferable to change the relative positions by
moving the two IR partial transmission filters in parallel, rather than
by adjusting the angle of the overlap.